Strain of Zoogloea adapt C-92

Use: in technology of wastewater treatment of city waste treatment facilities. Concept of the invention: new strain of Zoogloea adapt C-92 is suggested that was received by selective strain adaptation, derived from wastewater activated sludge of city wastewater treatment facilities. The strain is resistant against heavy metal ions, sorbs ions of heavy metals and uses petrochemicals as a carbon source. See Table 1.

Description of the invention.
The invention can be referred to industrial and ecological microbiology, biotechnology of waste treatment and concerns the new strain obtaining that produces exopolysaccharides, has high adsorptive capacity, is capable to flocculation and use of petrochemicals as a carbon source in aerotanks when recycling of city wastewater.

Use of microorganisms for wastewater treatment from the dissolved metals and recycling of organic impurities is known.

Strain of Cunnighamella blakesleeana is quite known which cell walls are capable to connect ions of copper (6.3 mm), cobalt (2.1 mm), zinc, iron and cadmium [1].
The defect of this strain is the necessity of preliminary biomass treatment for obtaining a fraction containing bacterial capsules, and also binding of metal is a temperature-dependent process having pH optimum that negatively affects when using it in wastewater treatment technology as the temperature in an aerotank is not stable, and pH varies 7 to 10.

Strain of Desulfovibrio desulfuricans is known for use under treatment of industrial wastewater from the dissolved metals by binding them in insoluble sulfides with hydrogen sulfide formed by bacteria [2].
The defect of this strain is the impossibility to use it in treatment of city waste water in aerotanks as it is a strict anaerobe and influence of toxic organic impurities on activity of hydrogen sulfide production is unknown.

Strain of Proteus mirabilis is known to be derived from activated sludge of waste treatment facilities and is capable to propagate in the presence of 300 mg/l of cadmium and to sorb it from the medium. Resting cells sorb up to 0,7 mg/l and growing ones - 0,22 mg/l of cadmium with concentration of 1 mg/l in the medium [3].
The defect of this strain is its specificity against cadmium ions. Besides presence of sum of heavy metal cations supposes to use bacteria which effectively sorb a number of cations. Cultural and biotechnological properties of the strain are not published.

The closest to the invention is strain of Zoogloea ramigera, synthesizing exopolysaccharides that act like polyelectrolytes and sorb cations of such metals as cobalt, copper, cadmium and iron. The sorbation reaches 95 % of the whole quantity of cations present in the solution at their concentration 12 to 117 mg/l [4]

The strain has the following defects: it is not adapted for working conditions in wastewater of aerotank containing a considerable quantity of toxic agents, requires preliminary biomass treatment that consists in immobilization of bacteria on the carrier calcium alginate, and also expensive media for its maintenance with vitamin B12 and amino acids - arginine and alanine.

The aim of the invention: obtaining of strain which produces exopolysaccharides, effectively sorbs ions of heavy metals in the conditions of aerotank wastewater abounding in the toxic impurities, is capable to use petrochemicals in wastewater as a carbon source, is unpretentious to nutrient mediums and dispenses with special equipment for cultivation.

The aim is reached by use of strain of Zoogloea adapt C-92 VKPM В-7040 that is a sorbent of heavy metal ions.
The suggested strain has been received by method of selective adaptation on the nutrient media containing salts of heavy metals, with the subsequent screening of the most stable forms.
The received strain of Zoogloea adapt C-92 was deposited by VKPM research institute of genetics on May 30, 1995 with registration number В-7040.
The strain was deposited with the scientific description (certificate).
The strain of Zoogloea adapt С-92 VKPM В-7040 is characterized by the following traits.
Morphological characters. Cells are rod-like, straight, 0.5 - 1.0 x 1.0 – 3.0 microns, mobile, in media with raised heavy metal salt content cells are rounded and smaller. Forming of cysts or spores does not occur. Aggregation into microscopic flocs floating freely is possible. Production of exopolysaccharides. The quantity of polymer and flocculation degree depends on the culture age and medium structure. Grams stain is gram-negative.

Cultural characters. The strain is undemanding to growth factors, grows well on usual nutrient media (dry nutrient agar, meat infusion agar, meat infusion broth). Forming of fine, round, nonpigmented, shining colonies with even edges on agar media. Optimal temperature of growth is 25-28C, pH 7.0-7.2. The culture is sustained by replanting on dense nutrient media and stored in a glycerine solution at 70C or cool dehumidified.

Physiological characters. Aerobic chemoorganotrophs; denitrification positive. Non-producing indole and hydrogen sulfide. Oxidase and catalase positive. Oxidation of glucose, rhamnose, saccharose and lactose.
For Zoogloea adapt C-92 cultivation М9 medium of the following structure is used, g/l: Na2HPO4 6; KH2PO4 3; NaCl 0,5; NH4Cl 1; distilled water, the rest with addition of 1.5 % glucose. Cultivation is conducted on temperature-controlled shakers or in fermenters at 28C and intensive aeration during 18-24 h.
Biomass output is 109 cfu/ml.

The strain of Zoogloea adapt C-92 VKMP В-7040 is identified by Bergeys Manual [6].
The determinative differences between the supposed strain and the prototype strain are effective sorption of heavy metal ions in aerotank conditions;
simple nutrient medium;
usual equipment used for cultivation of aerobic bacteria.

Since the suggested strain was obtained for the first time and not used in manufacturing technologies, we can make a conclusion on conformity of the suggested strain with the invention criteria of "novelty" and "inventive height".

Example 1. Production of strain of bacteria Zoogloea adapt C-92. The new strain of Zoogloea adapt C-92 is obtained by means of extraction from activated sludge of city wastewater treatment facilities and its subsequent selection on М9 medium saturated with copper ions. The bacteria are seeded into М9 medium with addition of copper in the form of CuSO4 in quantity of 120 mg/l, incubated at 28C with aeration for 15 days and then subcultured in the fresh medium with a higher concentration of copper. A number of bacterial subcultures make it possible to select microorganisms stable against 290 mg/l of copper. They possess the raised stability against ions of zinc, iron, lead and cobalt. The bacteria obtained effectively sorb ions of heavy metals. After incubation in no-copper media the bacteria still have the same behavior.

Example 2. Biomass production and sorption of copper ions by bacteria Zoogloea adapt C-92 in laboratory conditions. The biomass is obtained by cultivation of bacteria in a fermenter in the form of suspension on М9 medium with addition of 1.5 % sugar. The temperature of incubation is 28C, pH 7.0, growth occurs in aerobic conditions for 24 h. The biomass obtained is washed off the medium of the distilled water and precipitated by centrifugation during 10 minutes at 8 000 rpm. Copper is added in the form of CuSO4, sorption is carried out at 25C within 1 h. Residual copper is defined by method of Tikhomirova L.P. [5]. It was shown that 1011 bacteria sorb 0.56 mg of copper in non-growing conditions.

Example 3. Determination of stability of bacterium Zoogloea adapt С-92 against copper ions. Studying of bacterial stability against the raised content of copper ions is carried out on aerotank wastewater with content of (mg/l): iron 1.0; lead 0.012; copper 0.06; phenol 0.05; petrochemicals 1.0 with addition of 300 ml/l of М9 medium and additional copper in the form of CuSO4 in quantity of 78 mg/l. 0.2 l of the prepared medium is put in a flask of 0.5 l capacity and inoculated with 0.2 ml of bacterial suspension. The initial bacterial titre makes 2.7105 cfu/ml. Cultivation is carried out at 28C with aeration during 5 days. So concentration of bacteria has increased to 5.0106 cfu/ml that makes 15 % of the check variant, cultivated without additional copper.

Example 4. Epigenetical studying of strain of Zoogloea adapt C-92 in the presence of petrochemicals. For studying of the strain epigenetics on М9 medium in the presence of 5 % petrochemicals (solar oil, kerosene, used oils in the ratio 43:5:52) bacteria (3.6104 cfu/ml) are seeded into the medium and incubated for 5 days with aeration at 28C. Concentration of bacteria increases to 2.0107 cfu/ml, that corresponds to growth of bacteria on М9 medium in the presence of 0.2 % sugar.

Thus, the strain is obtained that possesses the following advantages as compared with the known one:
effective sorption of heavy metal ions in aerotank conditions;
ability of use petrochemicals as a carbon source;
simple nutrient medium;
usual equipment used for cultivation of aerobic bacteria.

1. Venkateswerlu G. Stotzky G. Appl. Microbiol. and Biotechnol. 1989 V. 31, N 5-6, p. 619-625.
2. The patent of the Russian Federation RU 201 78114. Strain of bacteria Desulfovidrio Desulfuricans used for industrial wastewater treatment from ions of heavy metals.
3. V. Finoli C. FEMS Microbiol. Ecol. 1991 V. 85, N 3, p. 183-191.
4. Sabine P. Kuhn. Pfister P.M. Appl. Microbiol. and Biotechnol. 1989 V. 31, p. 613-618.
5. Tikhomirova L. P. Water supply and sanitary engineering. 1994, N 11, p. 30.
6. Bergeys Short Manual of Determinative Bacteriology. Edited by J. Holt, M. Meer, 1980.

Formula of invention.
Strain of bacteria Zoogloea adapt С-92 VKMP В-7040 used as a sorbent of heavy metal ions.

Documentary list quoted in the search report: 1. Sabine P.Kuhn., Pfister P.M. Appl. Microbiol. and Biotechnol. 1989 v. 31, p. 613 - 618.
Applicant(s): the State Research Centre of Virology and Biotechnology VECTOR.
Author: Borisova O.D.; Gusev V. A; Pugachev V. G; Bozhko N.A.; Repin V. E.
Patent holder: the State Research Centre of Virology and Biotechnology VECTOR.